TROSY-HNCA

Fig. 3. HNCA (a) and two implementations of HNCA-TROSY (b-c) experiments for recording intraresidual HN(/), 15N(/), 13C"(i) and sequential 1 HN(7), l5N(/), 13Ca(i — 1) correlations in 13C/15N/2H labelled proteins. Narrow and wide bars correspond to 90° and 180° flip angles, respectively, applied with phase x unless otherwise indicated. Half-ellipse denotes water selective 90° pulse to obtain water-flip-back.88,89 All 90°...

The magnetization has now been successfully transferred from the HN spin to the intraresidual and sequential 13C spins or alternatively to the interresidue 13C spin either using HNCA-TROSY or HNCO-TROSY schemes, respectively. It is inevitable that the HNCO-TROSY spectrum cannot be used for the sequential assignment alone because it does not bridge two sequential N shifts through common carbonyl carbon frequency. The... 

In the alternative approach, the HN(i), 15N( j, 13C (i/i— 1) correlations in the HNCA-TROSY spectrum can be supplemented with the data from the HN(CO)CA-TROSY experiment72 73 yielding solely 11 IN(/), 15N( ), 13C (i- 1) correlations. To this end, the HNCO-TROSY experiment is extended with the 13C —> 13C INEPT step, which utilizes rather large (ca. 51-55 Hz) one-bond scalar coupling between the 13C and 13C spins in order to transfer magnetization from the 13C (< — 1) nucleus further to the 13C ( — 1) spin. 

Fig. 6. The efficiency of the coherence transfer, for the first increment, as a function of delay 2Ta for the HNCA-TROSY. The transfer amplitudes for the intraresidual (a) and sequential (b) cross peaks were calculated with the following parameters,...

Four-dimensional extension of the HNCA-TROSY scheme... 

The HNCA-TROSY experiment can be readily extended to correlate the 13C spin of the preceding residue with intraresidual 11 IN(/), 15N(i), and 13C° (/ ) frequencies. This kind of four-dimensional HNCO CA-TROSY experiment (Fig. 7) was recently introduced by Konrat et al.79 The coherence flows through the following pathway... 

Thus, the magnetization is transferred from the amide proton to the attached nitrogen and then simultaneously to the intra- and interresidual 13C spins and sequential 13C spin. The 13C chemical shift is labelled during /, and 13C frequency during t2. The desired coherence is transferred back to the amide proton in the identical but reverse coherence transfer pathway. The 15N chemical shift is frequency labelled during t3, and implemented into the 13C 15N back-INEPT step. The sensitivity of the HNCOmCA-TROSY experiment is excellent and nearly similar to HNCA-TROSY except for the inherent sensitivity loss by a factor of /2, arising from additional quadrature detection needed for 13C frequency discrimination in the fourth dimension. The excellent sensitivity is due to a very efficient coherence transfer pathway,... 

Magnetization is initially transferred from 1Hn(i) to 15N(/) spin. Unlike in the HNCA-TROSY scheme, the desired coherence is transferred from the 15N(i) spin to the 13C7(/ — 1) spin of the preceding residue. To this end, nearly uniform fNc( 15 Hz) scalar coupling is used. As 2/NC is negligibly small, the coherence is transferred exclusively to the 13C (/ — 1) nucleus. Finally, the 13C —13C INEPT is used to transfer magnetization from 13C (i— 1) to... 

Fig. 15. Coherence transfer efficiencies as a function of delay 2TC for the residues in a-helix and ffisheet in the sequential HNCA-TROSY scheme. Equation (8) is plotted using the following parameters T2 un-trosy = 50 ms, T2 o = 25 ms, 2Ta = 25 ms,...

Fig. 19. Pulse scheme of the MP-HNCA-TROSY experiment. Delay durations A = 1/(4/hn) 2T a = 27 ms 2Ta= 18-27 ms 2TN = 1/(2JNC-) <5 = gradient + field recovery delay 0 < k < Ta/t2,inax- Phase cycling scheme for the in-phase spectrum is 0i = y 02 = x, — x + States-TPPI 03 = x 0rec = x, — x 0 = y. For the antiphase spectrum, f is incremented by 90°. The intraresidual and sequential connectivities are distinguished from each other by recording the antiphase and in-phase data sets in an interleaved manner and subsequently adding and subtracting two data sets to yield two subspectra.

Fig. 20. Enlargement of the region from the MP-HNCA-TROSY spectrum.

Fig. 21. Schematic illustration of MP-HNCA-TROSY antiphase (a) and in-phase (b) spectra with long acquisition time in q. The corresponding subspectra are shown after addition of the antiphase and in-phase data sets (c) and after subtraction of the antiphase and in-phase data sets (d). Due to very small Vcc > the intraresidual cross peaks are almost entirely cancelled out from the antiphase spectrum (a). In the subspectra, the intraresidual cross peaks are shown as doublets, separated by 53 Hz splitting in Fi-dimension, whereas sequential cross peaks are shown as singlets, and they exhibit 53 Hz offset for the upheld and downfield components between the subspectra.

Although, the MP-HNCA-TROSY experiment alone can yield sequential assignment, it can be also used concomitantly with the HNCA-TROSY experiment. This strategy is explained later, but let us first focus on the coherence transfer efficiency of the MP-HNCA-TROSY experiment. The transfer functions for the antiphase experiment (the efficiency for the in-phase experiment is practically the same) are calculated according to Eqs. (10) and (11) for the intraresidual... 

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